Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa

Figures

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  Figure 1. Pathological findings in EV-A71-infected organotypic culture epidermal squamous cells. At 2 days post-infection (dpi), prepuce epidermal squamous cells showed focal necrosis and vacuolated cytoplasm (A, arrows) and localization of viral antigens in the same lesion (B, arrows) and antigens (C, arrows) and viral RNA in other lesions (D, arrow). A negative control for the immunohistochemistry procedure that uses anti-Japanese encephalitis virus instead of anti-EV-A71 antibodies is shown in E (same lesion as in A and (B). A positive tissue control using EV-A71-infected hamster skeletal muscle is shown in (F, arrows). Stains: Hematoxylin and eosin (A), immunohistochemistry with permanent red chromogen/hematoxylin (B,C,E,F), and in situ hybridization with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/hematoxylin (D). Original magnification: 20x objective (A,B,E), 40x objective (C,D,F). Scale bars: 30 μm (A,B,E), 15 μm (C,D,F).

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  Figure 2. Pathological findings in EV-A71-infected lip epidermal and oral mucosa squamous cells. At 3 days post-infection, lip epidermal squamous lesions demonstrated viral antigens (A, arrows) and viral RNA (B, arrows) in the same lesion. Similarly, viral antigens (C, arrow) and viral RNA (D, arrow) were detected in the same infected superficial oral squamous mucosal lesion. Stains: Immunohistochemistry with permanent red chromogen/hematoxylin (A,C), and in situ hybridization with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/hematoxylin (B,D). Original magnification: 40x objective (A,B), 60X objective (C,D). Scale bars: 15 μm (A,B), 10 μm (C,D).

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  Figure 3. Double immunofluorescence staining of EV-A71-infected human prepuce and lip epidermis, and primary epidermal keratinocytes. Viral antigens were found in cytoplasm of prepuce epidermal keratinocytes (A,C, arrows) which were uniformly cytokeratin AE1/3 positive (B). Viral antigens in cytoplasm of prepuce epidermal keratinocytes (D,F, arrows) in uniformly SCARB2-protein positive keratinocytes (E). Both AE1/3 and SCARB2 (B,C,E,F) were negative in the dermal layers (*). S100 protein-positive Langerhans cells (H, arrows) were found adjacent to keratinocytes with viral antigens (G,I, arrows). There was no apparent co-localization of viral antigens in Langerhans cells. The dermal layer (*) is negative for both S100 and viral antigens (H,I). Viral antigens were found in AE1/3-positive primary epidermal keratinocytes (J–L, arrows) and SCARB2-positive primary keratinocytes (M–O, arrows). Stains: Immunofluorescence staining with permanent red chromogen (A,D,G,J), anti-human cytokeratin AE1/3/IgG conjugated with Alexa Fluor 488 (B,K), SCARB2/IgG conjugated with Alexa Fluor 488 (E) and Alexa Fluor 594 (N), S100/IgG conjugated with Alexa Fluor 488 (H), and DAPI (4′,6-diamidino-2-phenylindole,…

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  Figure 4. Virus replication in (A) EV-A71-infected human prepuce skin at 2, 4 and 6 days post-infection (dpi), and (B) primary epidermal keratinocytes at 1, 2, 3 dpi. Viral titers are expressed as CCID50/ml. Combined viral titers from organotypic tissue homogenates (intracellular) and supernatant (extracellular) were statistically significant between 2 and 6 dpi (P = 0.001) and between 4 and 6 dpi (P = 0.005), respectively. Supernatant viral titers from primary epidermal keratinocytes (B) between all time points were significantly significant (P < 0.05).

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  Figure 5. Infection of primary epidermal keratinocytes. Cytopathic effect (A, arrows) at days post-infection. Infected keratinocytes stained by immunohistochemistry with 3, 3′ diaminobenzidinetetrahydrochloride chromogen/hematoxylin at low (B) and high magnifications (C, arrows). Original magnification: 10x objective (B), 40x objective (C). Scale bars: 50 μm (B), 15 μm (C).